Glycine-serine-rich effector PstGSRE4 in Puccinia striiformis f. sp. tritici inhibits the activity of copper zinc superoxide dismutase to modulate immunity in wheat

Puccinia striiformis f. sp. tritici (Pst) secretes an array of specific effector proteins to manipulate host immunity and promote pathogen colonization. In a previous study, we functionally characterized a glycine-serine-rich effector PstGSRE1 with a glycine-serine-rich motif (m9). However, the mechanisms of glycine-serine-rich effectors (GSREs) remain obscure. Here we report a new glycine-serine-rich effector, PstGSRE4, which has no m9-like motif but inhibits the enzyme activity of wheat copper zinc superoxide dismutase TaCZSOD2, which acts as a positive regulator of wheat resistance to Pst. By inhibiting the enzyme activity of TaCZSOD2, PstGSRE4 reduces H2O2 accumulation and HR areas to facilitate Pst infection. These findings provide new insights into the molecular mechanisms of GSREs of rust fungi in regulating plant immunity.


Author summary
Pst secretes numerous effectors to modulate host defense systems. However, the mechanisms of these effectors, especially for glycine-rich or serine-rich effectors, remain obscure. In this study, we identified a new glycine-serine-rich effector, PstGSRE4, which exhibits unusual biochemical properties and is highly induced during early stages of infection. Transgenic expression of PstGSRE4-RNAi constructs in wheat significantly reduced virulence of Pst and increased H 2 O 2 accumulation in wheat. Overexpression of PstGSRE4 in wheat significantly increased virulence of Pst and reduced H 2 O 2 accumulation in wheat. PstGSRE4 was shown to target the ROS-associated regulatory factor TaCZSOD2, which was proved as a positive regulator of wheat immunity in this study. Further study revealed that PstGSRE4 inhibited the enzyme activity of TaCZSOD2 and thus compromises the host immune systems. This work reveals a novel strategy that rust fungi exploit to modulate host defense and facilitate pathogen infection.

Introduction
In nature, plants are exposed to a variety of biotic and abiotic stresses, including the invasion of numerous pathogenic microorganisms. In their interactions, plants and pathogens confront processes of defense and pathogenicity and co-evolve. Upon pathogen infection, pattern recognition receptors (PRRs) in plants recognize the pathogen-associated molecular pattern (PAMP) and activate PAMP-triggered immunity (PTI) to form the first level of defense [1].
Pathogens have formed a large number of virulence factors during the long-term evolution with the host, and successfully infect and colonize the host by acting on the host plant cells [1]. Effectors, as a type of very important virulence factors, are secreted from the pathogen into the host primarily to inhibit the host's defense response, and thus cause host plant susceptibility.
In addition, when certain avirulence effectors from the pathogen are directly or indirectly recognized by plant disease-resistant proteins, the plant immune system is strongly activated to induce the host cell hypersensitive response (HR), which has been termed effector-triggered immunity (ETI) [2]. Therefore, the effectors of a pathogen have a dual function of virulence and avirulence, which is not only an important weapon of pathogenicity, but also an important target of the plant immune system. Both PTI and ETI include the induction of reactive oxygen species (ROS), a key component of the defense system [3,4]. The sharp increase of ROS is a common manifestation when plants are confronted with various pathogens, indicating that ROS play a vital role in the process of plant resistance to pathogens. ROS burst is generally defined as a rapid production of high levels of ROS in response to external stimuli [5]. Superoxide radicals (O 2 -) and hydrogen peroxide (H 2 O 2 ) are considered important ROS in response to biotic stress [6]. During plant-pathogen interaction, penetration of pathogen into host plasma membrane triggers the early O 2 − burst by an NADPH oxidase, then they are rapidly converted to H 2 O 2 by dismutation [5]. Most of the data seem to indicate that the major ROS building the oxidative burst is H 2  decreased the number of bacteria in Nicotiana benthamiana, and then the number of bacteria significantly increased following the addition of SOD or other reactive oxygen scavengers [7]. On the other hand, H 2 O 2 can also act as signaling molecules to directly or indirectly activate the expression of resistance genes and defense genes. H 2 O 2 can induce the increase of antioxidant enzyme activity in plants to resist the invasion of pathogens. Exogenous H 2 O 2 can induce a significant increase in glutathione S-transferase (GST) transcription in soybean suspension cells, and H 2 O 2 scavengers can prevent this effect [8]. In addition, H 2 O 2 also participate in the lignification of cell walls and the cross-linking of proteins to cell walls to strengthen plant cell walls against pathogen invasion. After infection with diseased substances, synthesis of H 2 O 2 was observed in lignification sites of plant tissues [7]. H 2 O 2 can also induce the occurrence of plant HR response. A large number of experiments proved that exogenous H 2 O 2 can induce HR in cells of Arabidopsis thaliana [9]. Interestingly, many studies have shown that effector proteins can control the host immune response by interfering with the host ROS signaling pathway [10][11][12][13]. Understanding the mechanism of effectors regulating ROS-related targets will increase our knowledge of molecular mechanisms underlying the interaction between plants and phytopathogens, and provide a theoretical foundation to achieve durable disease resistance. Superoxide dismutase (SOD) is an important component of the antioxidant enzyme system and is widely distributed in microorganisms, plants and animals. It catalyzes superoxide anion (O 2 − ) radical disproportionation to produce O 2 and H 2 O 2 , and plays an important role in the balance between oxidation and oxidation resistance [14]. Based on their metal cofactors, protein folds, and subcellular distribution, SODs are mainly categorized as CuZnSODs, FeS-ODs, and MnSODs [15]. A previous study indicated that infection of grape with grapevine fanleaf virus caused the accumulation of ROS and activated its enzyme defense system, including SOD [16]. Among the isoenzymes of SOD in sunflower, the expression of CuZnSOD under biological stress is the most affected, indicating that CuZnSOD is the main antioxidant defense enzyme [17]. When the CuZnSOD gene in tomato chloroplasts was transferred into two N. benthamiana strains, it enhanced the resistance to anthrax by changing the expression of the antioxidant enzyme [18]. In the Phaseolus vulgaris-Uromyces appendiculatus interaction, the expression of CuZnSOD was increased greatly during the incompatible interaction [19]. Recently, in the study of barley-powdery mildew interaction, loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of copper-zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in Rom1 re-established HvSOD1 levels [20]. In the study of rice-Magnaporthe oryzae interaction, different SODs in miR398b regulated resistance to rice blast disease, and miR398b increased total SOD activity to upregulate the H 2 O 2 concentration and thereby improve disease resistance [21]. However, there have been no reports on phytopathogenic effectors targeting and regulating CuZnSODs from plants to suppress host immune response.
Among the diseases caused by rust fungi, the diseases on Gramineae and Leguminosae seriously threaten the safety of food production in China and throughout the world [22]. Stripe rust is one of the most serious diseases of wheat in the world [23]. Wheat has evolved resistance genes to protect against disease. However, Pst constantly mutates to overcome these resistance genes, and the effectors contributed significantly to the virulence diversity of Pst [24]. Due to the importance of effector proteins in the interaction between pathogens and plants, more and more attention has been paid to the study of effector proteins. Recently, stripe rust effector Pst18363 has been reported to stabilize a negative regulator of wheat defense, TaNUDX23, which suppresses ROS accumulation and facilitates Pst infection [25]. Another stripe rust effector, Pst_12806, is translocated into chloroplasts and perturbs photosynthesis, avoiding triggering cell death and supporting pathogen survival on living plants [26]. In several organisms, glycine-or serine-rich proteins have been shown to participate in RNA splicing, metabolism and signal transduction [27,28]. Pathogen effectors with a high content of glycine or serine could potentially modify the host's metabolism or signal transduction [29]. In Pst, a glycine-serine-rich effector protein PstGSRE1 containing a glycine-serine-rich motif (m9) has been shown to disrupt the nuclear localization of TaLOL2 and suppress ROS-mediated cell death induced by TaLOL2, thus compromising host immunity [29]. However, the mechanisms of glycine-serine-rich effectors, remain obscure, and further investigation is required. In this study, we characterized a new glycine-serine-rich effector protein PSTCY32_07414 (alias PstGSRE4), which lacks the m9-like motif, targets a wheat copper zinc superoxide dismutase TaCZSOD2. PstGSRE4 is required for full virulence of Pst in wheat. Further analyses showed that TaCZSOD2 is a positive regulator of wheat resistance to Pst, and PstGSRE4 reduces H 2 O 2 accumulation by inhibiting the activity of TaCZSOD2 to facilitate Pst infection. Our results provide new insights into the molecular mechanisms of glycine-serine-rich effectors of rust fungi regulating host immunity.
which was enriched in glycine (12.93%) and serine (19.40%) and did not contain any known functional domains except for a 22 amino acid (aa) signal peptide at its N-terminus (S1A Fig).
BLASTp analyses revealed that homologs of PstGSRE4 can be found only in rust fungi, including 13 in Pst, seven in Puccinia triticina (Pt) and four in Puccinia graminis f. sp. tritici (Pgt) (S1 and S2 Tables), indicating that GSREs constitute a large family within the rust fungi. Using MEME, we found that PSTCYR32_07414 did not have the motif as the m9 motif of PstGSRE1 (S1B Fig). Moreover, yeast two-hybrid (Y2H) assay indicated that PstGSRE4 does not interact with TaLOL2 (S1C Fig), suggesting that it is functionally diverged. Thus, PSTCYR32_07414 was designated Puccinia striiformis Glycine-Serine-Rich Effector 4 (PstGSRE4) and selected for further study.

Relative transcript levels of PstGSRE4 at different Pst infection stages
In order to characterize the expression pattern of PstGSRE4 during Pst infection stages, we analyzed its relative transcript levels by qRT-PCR at different time points during the infection process. We used fresh ungerminated urediniospores of CYR32 and infected wheat tissues collected from 6 to 264 hpi to detect the transcripts of PstGSRE4. qRT-PCR assays showed that PstGSRE4 was upregulated during Pst infection (6-

PstGSRE4 suppresses Pst322-and Bax-induced cell death by decreasing H 2 O 2 accumulation
SignalP 5.0 analysis showed that PstGSRE4 has a signal peptide encoded by the first 22 amino acids. Secretion of PstGSRE4 was verified through a signal sequence trap system [29]. pSUC2T7M13ORI-PstGSRE4 was transferred into the yeast SUC2-minus strain, YTK12. The fusion of the signal peptide of PstGSRE4 to the mature sequence of SUC2 promoted the successful secretion of invertase, which enables the yeast cells to hydrolyze raffinose and grow on YPRAA media (S3 Fig). In addition, we found that the TTC-treated PstGSRE4 culture filtrates turned red, confirming invertase activity (S3 Fig). The oomycete effector Avr1b was used as positive control, and the YTK12 strains with or without pSUC2 vector were used as negative controls. These results indicate that the signal peptide of PstGSRE4 is functional.
To examine the function of PstGSRE4, we used agro-infiltration to transiently express it in N. benthamiana. We observed that PstGSRE4 inhibited an elicitor-like protein [28] Pst322-induced cell death (S4A Fig). In addition, PstGSRE4 suppressed the pro-apoptotic protein Baxinduced cell death (S4B Fig). Accumulation of PstGSRE4, Pst18363, eGFP, Pst322 and Bax proteins in infiltrated tissue were confirmed by Western blots (S4C Fig). Because

PstGSRE4 suppresses callose deposition and Pst-induced H 2 O 2 accumulation
The important function of fungal effectors is to suppress PTI and/or ETI in order to create a suitable environment for infection. To analyze the ability of PstGSRE4 to inhibit PAMP-triggered responses, we used the bacterial type III secretion assay [29] to deliver PstGSRE4 into wheat leaves (S6A Fig). Callose deposition triggered by Pseudomonas fluorescence strain EtHAn carrying pEDV6-PstGSRE4 was significantly reduced at 24 and 48 hpi compared with the control EtHAn with or without pEDV6-RFP (S6B and S6C Fig). We further analyzed H 2 O 2 accumulation triggered by Pst race CYR23 which is incompatible with Suwon11. The results showed that after transient transformation, H 2 O 2 accumulation was significantly suppressed with EtHAn-PstGSRE4 at 24 and 48 hpi compared with the control EtHAn with or without pEDV6-RFP (S6D and S6E Fig). These results indicated that delivering PstGSRE4 into wheat inhibits PTI-associated callose deposition as well as H 2 O 2 accumulation in wheat.

Silencing of PstGSRE4 reduces virulence of Pst
To test whether PstGSRE4 is involved in pathogenicity of Pst, host-induced gene silencing To further confirm the virulence function of PstGSRE4 in Pst infection using stable transgenic plants, we prepared the RNA interference (RNAi) construct pAHC25-PstGSRE4-RNAi (S9A Fig) and delivered it into wheat cv. XN1376 by particle bombardment. Two T 4 transgenic wheat lines (L19 and L76) containing pAHC25-PstGSRE4-RNAi displayed significantly enhanced resistance against the virulent Pst race CYR31 (Fig 1A). Transcript levels of PstGSRE4 during Pst infection of transgenic lines L19 and L76 were significantly reduced ( Fig  1B). The results showed that both lines L19 and L76 contained at least one copy of the transgene (S9B Fig). Compared with control plants, fungal biomass in infected leaves of transgenic lines L19 and L76 was significantly reduced (Fig 1C). We also detected the contents of H 2

Overexpression of PstGSRE4 reduces wheat resistance to Pst
To further confirm the virulence function of PstGSRE4 in Pst infection in stable transgenic plants, we prepared the overexpression construct pCAMBIA3301-PstGSRE4-overexpression (S10A Fig) and delivered it into wheat cv. Fielder by A. tumefaciens-mediated stable transformation. Two T 3 transgenic lines (L2 and L3) containing pCAMBIA3301-PstGSRE4-overexpression displayed significantly reduced wheat resistance against Pst race CYR23 (Fig 2A).
The results showed that both L2 and L3 contained at least one copy of the transgene (S10B and S10C Fig). RT-PCR (S10D Fig) and qRT-PCR (Fig 2B) showed that the expression of PstGSRE4 was different in each line, and it influenced the resistance against Pst race CYR23 to a different extent. Compared with control plants, fungal biomass in infected leaves of transgenic lines L2 and L3 was significantly increased (Fig 2C).

PstGSRE4 specifically targets wheat copper zinc superoxide dismutase TaCZSOD2
To understand the potential virulence function of PstGSRE4 in wheat, a yeast two-hybrid (Y2H) library was constructed to screen constructs and identify potential host targets of PstGSRE4. With PstGSRE4 (ΔSP) as the bait, several candidate targets were identified (S3 Table). We selected the candidate ROS-associated genes for further study. A candidate target sequence was annotated as superoxide dismutase [Cu-Zn] (TraesCS7A02G292100.1) (http:// plants.ensembl.org/index.html). According to a previous study, we collected 26 TaSODs, 8 AtSODs and 8 OsSODs from the Arabidopsis Information Resource (TAIR10) database (http://www.arabidopsis.org/index.jsp), the Rice Genome Annotation Project (RGAP) database (http://rice.plantbiology.msu.edu/) and the wheat reference genome IWGSC v1.1 (Evalue < 1e -5 ), respectively. A phylogenetic tree was constructed, which revealed that the CuZuSOD gene obtained in this study lies within the same clade as AtCSD2 (S11 Fig and S1 File). Based on this evidence, we designated the wheat CuZnSOD as TaCZSOD2. BlastN analyses of the wheat genome showed that there were three copies of TaCZOD2 located on chromosomes 7A, 7B and 7D, respectively. Sequence alignment showed that the three copies, designated as TaCZSOD2-7A, TaCZSOD2-7B and TaCZSOD2-7D, and the TaCZSOD2 obtained in this study share 99.3% nucleotide identity (S12 Fig). We cloned TaCZSOD2 and the other three TaCZSODs (TaCZSOD1, TaCZSOD3, TaCZSOD4) from wheat cultivar Suwon11 to further confirm the preliminary results of the Y2H assay (Fig 3A and 3B). The results showed that PstGSRE4 specifically interacts with TaCZSOD2.
To determine whether PstGSRE4 can directly interact with TaCZSOD2, we used recombinant proteins PstGSRE4-GST, TaCZSOD2-His and GST, TaCZSOD1-His (as negative control) expressed from E. coli BL21 to conduct GST pull-down assay (Fig 3C). We detected immunoprecipitated protein complexes by western blotting. TaCZSOD2-His was detected in PstGSRE4-GST pull-down fractions but not the TaCZSOD1-His, indicating that PstGSRE4 specifically interacts with TaCZSOD2 in vitro. To obtain further experimental evidence, we conducted a Co-IP experiment based on A. tumefaciens-mediated transient expression in N. benthamiana. PstGSRE4 mature protein fused to GFP and TaCZSOD2 or TaCZSOD1 fused to HA were co-expressed in N. benthamiana. Western blotting analysis showed that PstGSRE4

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat specifically interacts with TaCZSOD2 in vivo (Fig 3D). We also co-expressed PstGSRE4 and TaCZSOD2 in N. benthamiana cells and found that PstGSRE4 and TaCZSOD2 were co-localized in the cytoplasm (S13A and S13B Fig), suggesting that PstGSRE4 and TaCZSOD2 interact in the cytoplasm. To further confirm that PstGSRE4 and TaCZSOD2 interact in the cytoplasm, coding sequences of ΔTP-TaCZSOD2 (TaCZSOD2 without chloroplast transit peptide) was ligated into pBINGFP2 to construct the recombinant plasmid ΔTP-TaCZSOD2-GFP. And we co-expressed ΔTP-TaCZSOD2-GFP with PstGSRE4-RFP, TaCZSOD2-GFP with PstGSRE4-RFP, ΔTP-TaCZSOD2-GFP with RFP, and TaCZSOD2-GFP with RFP in N. benthamiana, and detected the activity of CuZnSOD. The results showed that TaCZSOD2 had enzyme activity in the cytoplasm, while PstGSRE4 inhibited the activity of TaCZSOD2 in the cytoplasm (S13C and S13D Fig). Compared with full length-TaCZSOD2, PstGSRE4 significantly inhibited the activity of ΔTP-TaCZSOD2 (S13C and S13D Fig).

TaCZSOD2 positively regulates wheat resistance against Pst
To confirm the function of TaCZSOD2 in wheat resistance to Pst, we knocked down expression of TaCZSOD2 in wheat leaves by BSMV-VIGS. Two specific fragments were designed for Only the yeast co-expressing PstGSRE4 and TaCZSOD2 grew on the medium SD-Trp-Leu-His-Ade and yielded X-α-gal activity. Co-expressing PstGSRE1 and TaCZSOD2 cannot grow on the medium SD-Trp-Leu-His-Ade. (B) PstGSRE4 specifically interacts with TaCZSOD2 in yeast. Only the yeast co-expressing PstGSRE4 and TaCZSOD2 grew on the medium SD-Trp-Leu-His-Ade and yielded X-α-gal activity. Co-expressing PstGSRE4 and TaCZSOD1, TaCZSOD3 or TaCZSOD4 cannot grow on the medium SD-Trp-Leu-His-Ade. (C) PstGSRE4 interacts with TaCZSOD2 in vitro. A GST pull-down assay was used to detect the interaction between PstGSRE4-GST and TaCZSOD2-His. TaCZSOD2-His and PstGSRE4-GST were detected with anti-His or anti-GST antibodies, respectively. TaCZSOD1-His was used as negative control. (D) PstGSRE4 interacts with TaCZSOD2 in vivo. Co-immunoprecipitation (IP) was performed on extracts of N. benthamiana leaves expressing both PstGSRE4-GFP and TaCZSOD2-HA. GFP was detected by western blot with anti-GFP antibodies. HA was detected by western blot with anti-HA antibodies. The TaCZSOD1-HA was used as negative control. https://doi.org/10.1371/journal.ppat.1010702.g003

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat silencing TaCZSOD2 (S14A Fig). qRT-PCR analysis showed that, during the interaction between wheat and Pst, transcript levels of TaCZSOD2 were up-regulated at 12, 48 and 96 hpi with the avirulent Pst race CYR23, and up-regulated at 96 and 120 hpi with the virulent Pst race CYR31 (S14B Fig). After virus inoculation, plants displayed mild chlorotic mosaic symptoms at 10 dpi. Compared with the control plants, fewer necrotic spots and sporadic uredia appeared on leaves of TaCZSOD2-knockdown plants after inoculation with the avirulent race CYR23, whereas no significant differences were found on leaves of TaCZSOD2-knockdown plants after inoculation with the virulent race CYR31 (Fig 4A). The silencing efficiency monitored by qRT-PCR indicated that TaCZSOD2-1as/2as transcript levels in knockdown plants were significantly reduced (Fig 4B). Moreover, the transcript levels of the other TaCZSODs were not influenced after the expression of TaCZSOD2 was knocked down (Fig 4C). The biomass of Pst/wheat showed an increase in the leaves inoculated with BSMV:TaCZSOD2 after inoculation with the avirulent race CYR23 compared with the leaves of BSMV:γ-inoculated wheat (Fig 4D). The biomass of Pst/wheat showed no significant changes in the leaves inoculated with BSMV:TaCZSOD2 after inoculation with the virulent race CYR31 compared with the leaves of BSMV:γ-inoculated wheat (S14C Fig). We detected the activity of CuZnSOD in TaCZSOD2-knockdown plants after inoculation with the avirulent race CYR23. Compared with the control plants, the enzyme activity of TaCZSOD2-knockdown plants was significantly reduced (Fig 4E) To further test the function of TaCZSOD2 in wheat defense against Pst, we also performed transient delivery of TaCZSOD2 using the bacterial T3SS system in wheat. At 48 h post-infiltration with a plasmid carrying EtHAn strains, wheat plants were inoculated with Pst virulent race CYR31. The number of uredia was significantly reduced at 14 dpi (S15A Fig). In addition, the area of H 2 O 2 accumulation and HR areas were significantly increased (S15B-S15D Fig).
To further confirm the function of TaCZSOD2 during Pst infection of stable transgenic plants, we prepared the overexpression construct CUB-TaCZSOD2-overexpression (S16A Fig) and delivered it into wheat cv. Fielder by A. tumefaciens-mediated stable transformation. Three T 2 transgenic lines (L1, L4 and L9) containing CUB-TaCZSOD2-overexpression displayed significantly increased wheat resistance against Pst race CYR31 (Fig 5A). PCR and Western blot assays indicated that L1, L4 and L9 contained at least one copy of the transgene (S16B and S16C Fig). qRT-PCR showed that the expression of TaCZSOD2 increased in each line (S16D Fig). And the fungal biomass in infected leaves of transgenic lines L1, L4 and L9 were significantly reduced (Fig 5B). Also, compared with control plants, the enzyme activities of CuZnSOD in transgenic lines L1, L4 and L9 were significantly increased (Fig 5C). We also detected the contents of H 2 O 2 and O 2 in infected leaves of TaCZSOD2-overexpression plants at 6, 12, 24 and 48 hpi (Figs 5D and S16E). The results showed that overexpression of TaCZ-SOD2 significantly increased H 2 O 2 accumulation and reduced O 2 − accumulation in wheat.
Histological changes in TaCZSOD2-overexpression plants infected with CYR31 were observed by microscopy. As shown in Figs 5E and S16F-S16H, H 2 O 2 accumulation and HR area triggered by Pst race CYR31 were significantly increased in TaCZSOD2-overexpression plants at 48 hpi/72 hpi with the increasing enzyme activity of CuZnSOD. These findings indicate that TaCZSOD2 positively regulates wheat resistance against Pst.

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat

TaCZSOD2 increases H 2 O 2 accumulation
In order to further confirm that TaCZSOD2 can increase H 2 O 2 accumulation, we use chitin to induce a rapid oxidative burst to determine the capacity of TaCZSOD2 to maintain redox balance in TaCZSOD2 In summary, these results suggested that TaCZSOD2 has the ability to increase H 2 O 2 accumulation.

PstGSRE4 inhibits the activity of TaCZSOD2
In order to confirm the effect of PstGSRE4 on the activity of TaCZSOD2, we used recombinant proteins PstGSRE4-His, TaCZSOD2-His and eGFP-His, TaCZSOD1-His (as negative control) expressed in E. coli BL21 to conduct enzyme activity assays by the NBT photoreduction method (Fig 6A and 6B). The results demonstrated that PstGSRE4 reduces the enzyme activity of TaCZSOD2 in vitro, but not TaCZSOD1. Meanwhile, we used agro-infiltration to transiently co-express PstGSRE4 and TaCZSODs in N. benthamiana to determine whether PstGSRE4 inhibits the activity of TaCZSOD2 (Fig 6C and 6D). The results showed that PstGSRE4 reduces the activity of TaCZSOD2 in vivo. To further explore the effect of PstGSRE4 on TaCZSOD2 in vivo, we also used PstGSRE4-overexpression transgenic lines L2 and L3 to detect the activity of CuZnSOD in wheat (Fig 6E and 6F). The results showed that the activity of CuZnSOD was reduced when the expression of PstGSRE4 increased. These results indicated that PstGSRE4 can reduce the enzyme activity of TaCZSOD2.

Discussion
Glycine-or serine-rich proteins perform important, even decisive roles during infection in various pathogens [27][28][29][30][31][32][33]. However, few glycine-serine-rich effectors (GSREs) have been characterized in Pst. In our previous study, we identified four glycine-serine-rich effectors in Pst. Among these candidates, we focused on PstGSRE1, which contains the m9 motif, and was found to target the ROS-associated transcription factor TaLOL2 [29]. Further sequence analysis of the GSREs revealed that only PstGSRE4 lacks the m9-like motif (S1B Fig), suggesting the functional divergence with other GSREs. Silencing PstGSRE4 decreased Pst growth and development (Figs 1 and S7 and S8), due to the increased ROS accumulation in wheat. ROS have been proposed to orchestrate the establishment of plant defenses following HR [3,34]. In plant cells, ROS has been identified as playing a key role in the development of HR and systemic immunity [35]. Inhibition of this reaction is an important strategy for the successful infection and colonization by obligate biotrophic pathogens. Overexpression of PstGSRE4 suppressed ROS accumulation induced by the avirulent Pst race CYR23 and the deposition of callose induced by EtHAn (S6 Fig),

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat PstGSRE4, Pst effector PstGSRE1 was also reported to be involved in suppression of callose deposition and ROS accumulation [29]. In M. oryzae, some glycine-or serine-rich effectors regulate the activity of a variety of antioxidant enzymes, then inhibit the level of ROS in the host, and finally decrease the host immune response [30,31]. Therefore, we speculated that GSRE proteins play an important role in regulating the host immune response, and they may specifically regulate the ROS signal transduction pathway of higher plants. However, although

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat PstGSRE4 contains 12.93% glycine and 19.40% serine, it does not interact with TaLOL2 (S1 Fig), suggesting that it regulates ROS signal pathway in wheat via a different mechanism.
In this study, we found that PstGSRE4 specially interacts with wheat copper zinc superoxide dismutase TaCZSOD2 (Fig 3), an important isoform of SOD in plants. The plant CuZnSOD isoenzymes differ in their subcellular location, either plastid or cytosolic. Distinct subcellular localization of the individual CuZnSOD isoforms indicates the necessity of superoxide removal within specialized cellular compartments [36]. In Arabidopsis, the three CuZnSOD isoforms (CSD1, CSD2, and CSD3) are localized in the cytoplasm and nucleus, chloroplast and peroxisome, respectively. However, in this study, we found that TaCZSOD2, the ortholog of CSD2 of Arabidopsis, is not only localized in the chloroplast, but also in the cytoplasm (S13A Fig). Overexpression of ΔTP-TaCZSOD2 in N. benthamiana improved the CuZnSOD enzyme activity, but slightly lower than overexpression of full length of TaCZSOD2 (S13C and S13D Fig), suggesting that TaCZSOD2 of the chloroplast can also be activated via the CCS-independent pathway when localized in cytoplasm like CSD2 in Arabidopsis [37]. Meanwhile, PstGSRE4 inhibits the CuZnSOD enzyme activity that is increased by overexpression of TaCZ-SOD2 or ΔTP-TaCZSOD2 in N. benthamiana (S13C and S13D Fig). Thus, our data suggest that TaCZSOD2 can simultaneously carry out the function of the enzyme in the cytoplasm and chloroplast. The mechanism by which PstGSRE4 interacts with TaCZSOD2 in the cytoplasm remains to be further investigated. In addition, PstGSRE4 interacts only with TaCZSOD2, suggesting that there is a specific recognition site between PstGSRE4 and TaCZSOD2, but that site remains to be identified. Moreover, like rice CSD2 [21], silencing of TaCZSOD2 resulted in reduced CuZnSOD enzyme activity (Fig 4E), whereas overexpression of TaCZSOD2 led to higher CuZnSOD enzyme activity (Fig 5C), suggesting that TaCZSOD2 is an important CuZnSOD enzyme and positively contributes to CuZnSOD activity. In the process of plant-pathogen interaction, the activity of superoxide dismutase (SOD) positively or negatively correlates with plant disease resistance [38][39][40][41], this may be because each pathogen has its own unique mechanism of pathogenicity. However, no reports define the function of CuZnSOD in the interaction between wheat and rust fungi. In our study, silencing TaCZSOD2 in wheat reduced the H 2 O 2 accumulation triggered by the avirulent race CYR23 (Figs 4F and S14E and S14F). Meanwhile, compared with the control plants there were fewer necrotic spots and only sporadic uredia on TaCZSOD2-knockdown plants (Fig 4A). Moreover, bacterial delivery of TaCZSOD2 into wheat tissue (S15B and S15D Fig) and TaCZSOD2-overexpression lines (Figs 5D and S16F and S16H) increased wheat resistance to Pst in a ROS-dependent manner. Thus, our results indicated that TaCZSOD2 is a positive regulator of wheat resistance to Pst by increasing the accumulation of H 2 O 2 in wheat.
H 2 O 2 acts as a signal molecule to trigger resistance to various biotic and abiotic stresses [42][43][44]. Therefore, the pathogens usually secrete effectors to inhibit H 2 O 2 accumulation [45,46]. Moreover, H 2 O 2 also functions as intracellular and intercellular signal molecules to amplify the cellular ROS signals and trigger the HR [47][48][49][50]. HR is very effective against obligate biotrophic pathogens. During the interaction between wheat and Pst, the early burst of O 2 − may be induced by the contact of surface structures of the haustorial initials with the plasma membrane of mesophyll cells, whereas H 2 O 2 is induced to activate HR and other resistance responses [6]. In our study, PstGSRE4 inhibited the activity of TaCZSOD2 in vitro and in vivo (Fig 6), a detrimental response for wheat to accumulate more H 2 O 2 and successfully resist Pst. Meanwhile, the conclusion is confirmed again by the results that H 2 O 2 and HR induced by CYR23 were reduced in PstGSRE4-overexpression transgenic lines (Figs 2D and  2E, and S10F-S10H) and in TaCZSOD2-knockdown plants (Figs 4F and 4G, and S14D-S14G), along with the increase of O 2 − level. Thus, our results proved that PstGSRE4 inhibits the activity of total CuZnSOD by inhibiting the enzyme activity of TaCZSOD2 to control moderate H 2 O 2 accumulation upon Pst infection, and further promote stripe rust disease (Fig 7).

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat In this study, we also identified other three highly conversed TaCZSODs, TaCZSOD1, TaCZ-SOD3, and TaCZSOD4. The enzyme activity of TaCZSOD1 has been also detected in vitro (Fig 6A and 6B), suggesting that TaCZSOD1 may catalyze the production of H 2 O 2 from O 2 during Pst infection. Future work will be performed to investigate the functions of the three TaCZSODs during wheat-Pst interaction and reveal the molecular mechanisms of other Pst effectors targeting the CuZnSODs involved in wheat immunity. In summary, we identified a new glycine-serine-rich effector protein lacking the m9-like motif, PstGSRE4, which inhibits the enzyme activity of the wheat TaCZSOD2 to modulate ROSassociated defense responses. To our knowledge, this is the first direct evidence demonstrating that an effector in phytopathogens regulates the activity of CuZnSOD isoenzymes to suppress plant immunity. Previous studies indicated that CSD genes (mainly CSD1 and CSD2) from wheat and rice also play a positive role during abiotic stress [51,52]. Future studies will focus on using gene editing or overexpression technology to obtain broad-spectrum disease resistance and abiotic stress tolerance materials to advance novel strategies for protecting the wheat crop.

Plant materials and fungal Strains
In this study, we used wheat cultivar Suwon11 (Su11), Fielder and N. benthamiana. Wheat cultivar Su11 is highly susceptible to CYR31 and CYR32 and highly resistant to CYR23 [53]. Wheat seedlings were planted, inoculated with Pst and maintained in accordance with the procedures and conditions described previously [54]. Wheat cultivar Fielder was used for

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat transgenic transformation. Co-immunoprecipitation (CoIP) analysis was performed in fourleaf tobacco seedlings permeated with agrobacterium tumefaciens GV3101.
Freshly collected urediniospores of Pst race CYR23 were obtained from the leaves of wheat cultivar Thatcher (MX169), while CYR31 and CYR32 were obtained from Suwon11. The wheat cultivars Su11 and MX169 were grown at 16˚C in an artificial climate chamber.

Plasmid construction
The PstGSRE4 gene was cloned using complementary DNA from Pst CYR32. Full-length TaCZSOD genes were cloned from Su11. The amplicons were prepared using the appropriate restriction enzymes (S4 Table) and ligated into pBINGFP2 (a plasmid containing green fluorescent protein, GFP) for transient expression in tobacco, and pEDV6 for transient expression in wheat as well as pSUC2T7M13ORI (pSUC2), pEGX4T-1-GST, pET32a-His with the Clo-nExpress II One Step Cloning Kit (Vazyme Biotechnology, Nanjing, China). For VIGS analysis, specific cDNA segments of PstGSRE4 and TaCZSODs were predicted by siRNA finder software Si-Fi and then inserted into BSMV-γ carriers with NotI and PacI restriction sites [55]. For Co-IP assays, coding sequences of PstGSRE4 were ligated into pBINGFP2, and TaCZSOD2 and TaCZSOD1 were ligated into pICH86988 (a plasmid containing HA-tag), respectively. For Y2H assays, the coding sequences of PstGSRE4 and TaCZSODs were separately prepared using appropriate restriction enzymes (S4 Table) and ligated into pGADT7 and pGBKT7 vectors. The PstGSRE4 was ligated into pCAMBIA3301 for overexpression in wheat by Agrobacteriummediated transformation, and the TaCZSOD2 was ligated into CUB for overexpression in wheat by Agrobacterium-mediated transformation.

qRT-PCR analysis
To assay expression levels of PstGSRE4, urediniospores and leaves of wheat Suwon11 infected with CYR32 at 6,12,18,24,36,48,72,120,168,216 Table. Real-time PCR data were analyzed by the comparative 2 -ΔΔCT method to quantify relative gene expression [25]. The expression levels of PstGSRE4 and TaCZSOD2 were normalized to PstEF1 and TaEF-1α, respectively. Each sample was analyzed in three biological replications, and each PCR analysis included three technical repeats. The statistical significance was evaluated by Student's t-test.

Yeast signal sequence trap system
To validate the function of the predicted signal peptide of PstGSRE4, the yeast signal sequence trap system was used as described previously [56]. The predicted signal peptide sequence of PstGSRE4 was cloned into vector pSUC2T7M13ORI (pSUC2) using the specific primers (S4 Table) and then transformed into the invertase mutant yeast strain YTK12 [57]. To test the secretion function of the recombinant plasmid, positive clones were selected from the CMD-W medium then transferred to YPRAA medium to determine whether the recombinant plasmid had secretory function. In addition, invertase enzymatic activity was detected by the

Agrobacterium tumefaciens infiltration assays
The sequence encoding PstGSRE4 without the signal peptide (PstGSRE4(ΔSP)) was ligated into pGR107 carrier to construct the agrobacterium recombinant plasmid PVX-PstGSRE4-HA. The Avr1b gene from Phytophthora sojae and eGFP-HA were used as controls (S4 Table). A. tumefaciens cultures were prepared as described previously [59]. Resuspended A. tumefaciens cultures carrying each effector gene or eGFP at a final OD 600 of 0.2 and 10 mM MgCl 2 buffer were infiltrated into the leaves of 4-week-old N. benthamiana using a syringe without a needle. After 24 h, A. tumefaciens cultures for delivery of Bax or Pst322 at a final OD 600 of 0.2 were also infiltrated into the same site of N. benthamiana leaves. Expression of genes in all infiltration sites was detected by immunoblot three days after infiltration. Symptoms were monitored and recorded from 3 to 8 d after infiltration. Three independent biological replicates were conducted for each experiment.

Yeast two hybrid (Y2H) assay
TaCZSODs was constructed into pGBKT7 as bait, while PstGSRE4(ΔSP) was constructed into pGADT7 as prey (S4 Table). Then they were co-transformed into yeast strain AH109, plated on SD-Trp-Leu and SD-Trp-Leu-His medium, and cultured at 30˚C for 3 to 5 d. The monoclonals grown on SD-Trp-Leu-His were selected and diluted with water, then the interactions were confirmed by growth on the SD-Trp-Leu-His-Ade medium containing X-α-gal.

Bacterial T3SS-mediated overexpression in wheat plants
pEDV6-PstGSRE4(ΔSP), pEDV6-TaCZSOD2 were transformed into P. fluorescens strain EtHAn by electroporation. pEDV6-RFP was used as a control. Infiltration into wheat leaves was performed according to the method described previously [60]. The involvement in Pst pathogenicity or host defense response was tested by challenging the second leaves in pED-V6-PstGSRE4-inoculated wheat plants with Pst avirulent race CYR23 after 24 h. For determination of H 2 O 2 measurements, according to the previously described method [61], the inoculated leaves were sampled at 24 and 48 hpi and determined by 3-3'diaminobenzidine (DAB) staining. To examine the suppression of callose deposition, pEDV6-, pED-V6-PstGSRE4-and pEDV6-RFP-inoculated wheat plants were sampled at 48 hpi. Leaf samples were stained with 0.05% aniline blue in 67 mM K 2 HPO 4 (pH 9.0) overnight in darkness [29]. Leaves were rinsed in water and mounted in 50% glycerol and examined under an Olympus BX-53 fluorescence microscope (Olympus Corporation, Tokyo, Japan) using a DAPI filter. Images were acquired using a constant setting with 1000-ms exposure time. The number of callose deposits was quantified using ImageJ software [62].

Glutathione S-transferase (GST) pull-down assay
PstGSRE4(ΔSP) and TaCZSOD2/TaCZSOD1 were separately ligated into pGEX-4T-1 and pET22b/pET32a through enzyme digestion and ligation. Vectors were transformed into E. coli BL21 cells for protein expression. The corresponding protein was expressed and purified according to the prokaryotic expression procedure. GST-pull down kit (Thermo, Shanghai, China, UB281159) was used to validate the protein interactions in vitro. Another protein was detected by Western blot analysis. Horseradish peroxidase (HRP)-conjugated anti GST-Tag

Co-immunoprecipitation assays
PstGSRE4(ΔSP) and TaCZSOD2/TaCZSOD1 were ligated into pBINGFP2 and pICH86988 carriers, respectively. In addition, agrobacterium-mediated transient gene expression technology was used to co-express the above combinations in N. benthamiana. At 48 h after agroinfiltration, 100 μL of co-injected leaf proteins were extracted as the control (Input). Twenty μL of GFP Trap beads were added to the remaining extracts and incubated for 1 h, and centrifuged at 12000g at 4˚C for 1min. After removal of the supernatant, the beads in 60 uL volume of wash buffer were mixed with 20 uL of loading buffer, and heated at 100˚C for 5min. Precipitated proteins and crude proteins (Input) were detected by immunoblotting with an anti-GFP antibody (#A02020; Abbkine, Wuhan, China) and an anti-HA antibody (Beyotime, AF5057).

Activity assays of CuZnSOD
PstGSRE4-15bs, TaCZSOD2-15bs/TaCZSOD1-15bs and GFP-15bs were expressed in vitro by a prokaryotic expression system, then diluted to the same concentration after purifying by His-tag Purification Resin (BeyoGold, P2210) according to the protocol of manufacturer. The activity of TaCZSOD2/TaCZSOD1 was determined by nitroblue tetrazolium (NBT) reaction [63] in different combinations. The 3 mL reaction mixture contained 39 mM L-methionine 1.5 mL, 225 μM nitroblue tetrazolium (NBT) 0.3 mL, 8 μM riboflavin (dissolve in 30 μM EDTA-Na 2 buffer) 0.3 mL, 10 μL purified enzyme and 50 mM potassium phosphate buffer (pH 7.8) 890 μL. The reaction was initiated by illuminating the reaction mixture for 20 min, and photochemically produced superoxide reacted with NBT. Absorbance of formazan, the product of NBT reduction, was then recorded at 560 nm. One unit of SOD activity was defined as the amount of enzyme that caused 50% of the maximum inhibition of NBT reduction. These experiments were repeated three times. A standard curve of protein concentration was obtained with bovine serum albumin as standard [64].
In vivo, we determined the activity of CuZnSOD by using the CuZnSOD assay kit (colorimetry) (Jian Cheng, Nanjing, China, A001-4-1) according to the protocol of manufacturer. Weigh 0.2 g plant tissue sample accurately, add 4 times volume homogenate medium according to mass (g)-volume(ml) ratio of 1:4, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 3500 rpm for 10 min, take supernatant for assay. Take 0.1 ml 20% homogenate supernatant, add 0.2 ml homogenate medium (equals to 3 times dilution), mix sufficiently, take 3 samples of different volumes (10 μl, 30 μl, 50 μl), do pre-test according to operation table in order to determine optimal sample volume. Curve appears direct proportion while inhibition percentage is between 15-55%. Take the tube which inhibition percentage is between 45% to 50% as optimal sample volume. Use xanthine and xanthine oxidase reaction system to produce superoxide anion radicals (O 2 − ), the latter will oxidate hydroxylamine to form nitrite, appears prunosus color under effect of chromogenic agent, its absorbance can be measured by visible range spectrophotometer. If sample to assay contains SOD, then it has a narrow spectrum depressant effect for superoxide anion radicals, as result, absorbance in sample tube will be lower than absorbance in contrast tube, SOD activity can be calculated by formula. MnSOD and FeSOD loss activity in pretreated samples and CuZnSOD activity keeps stable. These experiments were repeated three times.

Barley stripe mosaic virus (BSMV)-mediated silencing
Based on the cloned PstGSRE4 and TaCZSOD2 genes, non-conserved regions were analyzed, and Premier Primer 5.0 was used to design gene silencing vector primers. According to

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat previously described methods [65], two fragments of PstGSRE4 or TaCZSOD2 were cloned and inserted into BSMV to produce BSMV:PstGSRE4-1/2as, BSMV:TaCZSOD2-1/2as. The wheat phytoene desaturase gene (PDS) was silenced as a positive control. BSMV:α and BSMV: β were mixed with BSMV:γ or recombinant γ-gene, in 1:1:1, and then the appropriate amount of FES buffer (2.613g dipotassium phosphate, 1.877 g glycine, 0.5 g sodium pyrophosphate, 0.5 g diatomite, 0.5 g porphyritic soil, 50 ml constant volume, 20 min sterilization by autoclaving) was added. Each independent experiment set FES buffer as a negative control, BSMV:γ as a blank control and BSMV:γ-TaPDS as positive controls for about 10 d to observe the symptoms of virus infection. After 10 to 14 d following inoculation, Pst races CYR23 and CYR31 (fresh urediniospores were collected from the infected leaves of Su11 that were grown at 16˚C in artificial climate chamber) were separately inoculated on the fourth leaf of wheat plants, which were placed in a dark and high humidity environment at 12˚C for 24 h, then grown in a normal 16/8 h light-dark cycle. The fourth leaves were sampled at 24, 48 or 120 hpi for assessment of silencing efficiency and histological observation. The phenotypes of the fourth leaves were photographed at 12 d after inoculation with Pst. These experiments were repeated three times.

Cytological observations of fungal growth and host response
The observation of necrotic death area hyphae and H 2 O 2 detection assay were performed as previously described [26]. Leaf segments were fixed and decolorized in a mixture of acetic acid/ethanol (1:1) for 3 d. Autofluorescence of mesophyll cells was observed to determine necrotic death area using epifluorescence microscopy (excitation filter, 485 nm; dichromic mirror, 510 nm; barrier filter, 520 nm). H 2 O 2 accumulation was detected by staining with DAB (Amresco, Solon, OH, USA). Hyphae were stained with WGA conjugated to Alexa-488 (Invitrogen, Carlsbad, CA, USA) and observed under blue-light excitation (excitation wavelength 450-480 nm, emission wavelength 515 nm). Only the site where an appressorium had formed over a stoma was considered to be a successful penetration. The H 2 O 2 accumulation, necrotic areas, hyphal length, and hyphal areas were observed with a BX-53 microscope (Olympus) and calculated using DP-BSW software.

Determination of the accumulation of O 2 − and H 2 O 2
In vivo, we determined the content of O 2 − by using the O 2 − assay kit (SA-2-G, Comin Biotechnology, Suzhou, China) according to the protocol of manufacturer. Weigh 0.1 g inoculated leaves in different hours accurately, add 10 times 65 mM phosphate buffer (pH 7.8) according to mass (g)-volume(mL) ratio of 1:10, cut tissue to small pieces, make homogenate in icewater bath. Centrifugate at 10000 g for 20 min, take supernatant for assay. The 900 μL reaction mixture contained 0.5 mL homogenate, 0.4 mL 10 mM hydroxylamine solution, 37˚C for 20

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat min. Then add 0.3 mL 17mM 4-aminobenzenesulfonic acid and 0.3 mL 7mM α-naphthylamine, 37˚C for 20 min. Then add 0.5 mL 1 chloroform, centrifugate at 8000 g for 5 min, take 1 mL supernatant for assay. Recorded absorbance at 530 nm against a distilled water blank. We determined the content of H 2 O 2 by using the H 2 O 2 assay kit (A064-1-1, Comin Biotechnology, Suzhou, China) according to the protocol of manufacturer. Weigh 0.1 g inoculated leaves in different hours accurately, add 10 times propanone according to mass (g)-volume (mL) ratio of 1:10, cut tissue to small pieces, make homogenate in ice-water bath. Centrifugate at 8000 g for 10 min, take supernatant for assay. The 1.3 mL reaction mixture contained 1 mL homogenate, 0.1 mL titanic sulfate solution and 0.2 mL ammonium water, centrifugate at 8000 g for 5 min, take sediment for assay. Then add 1 mL sulphuric acid solution to dissolve the sediment, let stand for 10 min at the room temperature. Recorded absorbance at 415 nm against a distilled water blank. These experiments were repeated three times.

Oxidative burst measurement
Leaves from 6-wk-old WT and TaCZSOD2-knockdown or TaCZSOD2-overexpression transgenic lines were sliced into 10 mm 2 discs, and maintained overnight in water in a 96-well plate. Then, the leaf discs were treated with 200 μL of solution containing 8nM chitin (hexa-Nacetyl-chitohexaose), 20 μg/ml peroxidase (Sigma-Aldrich) and 20 nM luminol. Luminescence was recorded for 30 min using a multiscan spectrum. Each data point consisted of six replicates. These experiments were repeated three times.

Statistical analyses
Statistical analyses of each treatment were performed with the statistical software version package of IBM SPSS Statistics 21 (IBM SPSS Statistics, IBM Corporation, Armonk, New York, USA).

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat time points according to the infection stage of Pst. US (Urediniospores) was used as a control. Relative transcript levels of PstGSRE4 were calculated by the comparative threshold (2 -ΔΔCT ) method. The quantitative RT-PCR values were normalized to the expression level for PstEF-1. The transcript level of PstGSRE4 at US stage was standardized as 1. Values represent the means ± SE of three independent replicates. Differences between time-course points were assessed using Student's t-test. Double asterisks indicate P < 0.01. (TIF) S3 Fig. PstGSRE4 has functional signal peptide. Functional validation of the putative N-terminal signal peptide of PstGSRE4 using the yeast invertase secretion assay. Yeast YTK12 strains carrying pSUC2-SP (Avr1b) and pSUC2-SP (PstGSRE4), which express two different signal peptides fused in frame to the mature invertase gene SUC2, were able to grow in YPRAA (Yeast-Peptone-Raffinose-Antimycin A) medium with raffinose as sole carbon source. YTK12 or YTK12 strains carrying empty vector pSUC2T7M13ORI were used as negative control. Invertase activity was detected with 2,3,5-triphenyltetrazolium chloride (TTC). The red color indicates invertase activity.

PLOS PATHOGENS
PstGSRE4 targets TaCZSOD2 to modulate immunity in wheat with EtHAn, EtHAn pEDV6-RFP, or EtHAn pEDV6-PstGSRE4 at 24 and 48 hpi with Pst. SV, substomatal vesicle. Tissues were stained with DAB. Scale bars, 20 μm. The amount of H 2 O 2 production was measured by calculating the DAB-stained area at each infection site using DP-BSW software. Values represent the means ± SE (n = 30). Asterisks indicate a significant difference (P < 0.05) relative to the control sample according to Student's t-test, double asterisks indicate P < 0.01.